Abstract

Mutations in SOD1 cause hereditary variants of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous, with toxicity deriving also from glia. In particular, microglia contribute to disease progression. Methylene blue (MB) inhibits the effect of nitric oxide, which mediates microglial responses to injury. In vivo 2P-LSM imaging was performed in ALS-linked transgenic SOD1(G93A) mice to investigate the effect of MB on microglia-mediated inflammation in the spinal cord. Local superfusion of the lateral spinal cord with MB inhibited the microglial reaction directed at a laser-induced axon transection in control and SOD1(G93A) mice. In vitro, MB at high concentrations inhibited cytokine and chemokine release from microglia of control and advanced clinical SOD1(G93A) mice. Systemic MB-treatment of SOD1(G93A) mice at early preclinical stages significantly delayed disease onset and motor dysfunction. However, an increase of MB dose had no additional effect on disease progression; this was unexpected in view of the local anti-inflammatory effects. Furthermore, in vivo imaging of systemically MB-treated mice also showed no alterations of microglia activity in response to local lesions. Thus although systemic MB treatment had no effect on microgliosis, instead, its use revealed an important influence on motor neuron survival as indicated by an increased number of lumbar anterior horn neurons present at the time of disease onset. Thus, potentially beneficial effects of locally applied MB on inflammatory events contributing to disease progression could not be reproduced in SOD1(G93A) mice via systemic administration, whereas systemic MB application delayed disease onset via neuroprotection.

Inhibition of microglial reaction towards axonal injury in the lateral column by local application of MB in vivo.

Microglial reaction towards laser-induced axonal transections within the lateral column of the spinal cord was recorded. Tissue injuries were induced by high-power laser pulses. The experiments have been performed in double transgenic mice expressing EGFP in microglia and EYFP in projection neurons. For better visualization, EYFP fluorescence in the images is depicted with a red colour table. Images are arranged such that rostral is to the upper side. (A-D) Left images were taken immediately (3 min) after axonal transection (autofluorescence and arrow) in control and mutant (SOD1G93A) mice. Respective images (right) were taken 60 min after injury. In respective experiments spinal cord was superfused with MB (1 mM). (E) Quantification of microglial response (increase of EGFP fluorescence around the injury) to the injured site. Control and mutant mice were of corresponding age (60 to 90 days of age). n = 7 mice for control response, n = 4 for MB-modified response in control mice, n = 9 for response in SOD1G93A mice, n = 7 for MB-modified response in SOD1G93A mice. (F) Breeding strategy to obtain SOD1G93A mice with fluorescently labeled microglia and projection neurons. Values are presented as mean ± SEM; ANOVA followed by Tukey test (*p<0.05, **p<0.01, ***p<0.001).

(A) Control and MB-treated (10 mg intraperitoneal injection per kg body weight per day) SOD1G93A mice with respect to the different stages of disease. (B and C) Weight and survival profiles for SOD1G93A mice (control and MB-treated). In B the data are presented relative to the highest value in each group. Values are presented as mean ± SEM; both groups: n = 16 mice; ANOVA followed by Tukey test (*p<0.05, ***p<0.001).

Rotarod motor performance of untreated and MB-treated (10 mg intraperitoneal injection per kg body weight per day) SOD1G93A mice. The time until mice fell off the rotarod at 12 rpm (A) and the velocity the mice reached using an acceleration rate of 1 rpm every 10 s (B) are presented. Each animal was tested three times per trial. Values are presented as mean ± SEM; both groups: n = 10 mice; ANOVA followed by Tukey test (*p<0.05, **p<0.01).

Microglial reaction towards laser-induced axonal transections within the lateral column of the spinal cord was recorded. Images are arranged such that rostral is to the left side. (A) Left images were taken immediately (5 min) after axonal transection (autofluorescence and arrow) in mutant (SOD1G93A) mice. Respective images (middle and right) were taken 30 and 60 min after injury, respectively. Upper images represent in vivo recordings of an exemplary non-treated SOD1G93A mouse. In the lower experiment, the mouse was treated with MB (10 mg oral per kg body weight per day; drug administration started at the age of 45 days). (B) Quantification of microglial response to the injured site. No significant differences in microglial reaction towards a laser-induced axonal injury were observed between non-treated and MB-treated SOD1G93A mice. Mutant mice were 60 to 90 days of age. Values are presented as mean ± SEM; no treatment: n = 9 mice, MB-treatment: n = 8.

(A) NeuN-stained cross-sections of the spinal cord at the level L3 to L5. Non-treated controls (SOD1G93A mice) are depicted left. Corresponding sections of MB-treated SOD1G93A mice are shown on the right (10 mg oral per kg body weight per day; drug administration started at the age of 45 days) (B) Counting of neurons in the anterior horn of SOD1G93A mice at different disease stages. Cell somata bigger than 20 µm were counted and given per mm2. In preclinical stages, neuron number was significantly higher in MB-treated mice compared to non-treated mice indicating an early neuroprotective effect of MB. Note that no differences were observed in later disease stages. Values are presented as mean ± SEM; n = 4 mice for each group; ANOVA followed by Tukey test (*p<0.05).

Effect of systemic application of MB on intracellular TDP-43-containing aggregates.

To analyze intracellular aggregations in anterior horn neurons, TDP-43 and SMI-32 staining was performed in lumbar cross-sections. TDP-43-containing aggregates in SMI-32-positive neurons were observed in the anterior horn of control and MB-treated pre-clinical and advanced clinical mice. An exemplary TDP-43-stained neuron from each group, marked by an arrow, is shown in the last line of images.